There is a continuing need in medical practice and research, and in analytical and diagnostic procedures, for rapid and accurate determinations of chemical and biological substances present in various fluids, such as biological fluids. For example, the presence of drugs, narcotics, hormones, proteins, toxins, microorganisms, viruses, steroids or nucleic acids must be rapidly and accurately detected for effective research, diagnosis or treatment of various diseases or conditions.
Prostaglandin E.sub.2 (PGE.sub.2) is a potent biochemical mediator of inflammation in the body. Elevated levels of PGE.sub.2 in crevicular fluid have been shown to be an indicator of periodontal disease. Due to the cyclical nature of periodontal diseases, an accurate measure of disease activity would be useful to determine periods of disease exacerbation and to aid in favorable disease treatment. If PGE.sub.2 can be detected at very low concentrations (100 nmolar or less), the clinical usefulness of PGE.sub.2 as an indicator of periodontal disease is optimized.
A wide variety of analytical methods have been developed in recent decades to detect various chemical or biological substances. Most of such methods rely on what are known in the art as "specific binding" reactions in which an unknown substance to be detected (known as a "specific binding ligand") reacts specifically and preferentially with a corresponding "receptor" molecule. Most well known specific binding reactions occur between immunoreactants, such as antibodies and antigens (foreign substances which produce immunological responses), but other specific binding reactions (such as avidin with biotin or a sugar with a lectin) are also known.
Many of the assay formats known in the art require one or more of the reactants to be immobilized on a solid substrate so that nonimmobilized reactants can be separated from immobilized reactants. "Sandwich" and "direct binding assays" are some of the assay formats which have been used in the art which require separation steps.
The standard immunological techniques, however, are more difficult to use with success in the detection of low molecular weight target specific binding ligands, such as PGE.sub.2. It is particularly desirable to detect low concentrations (1 nmolar or less) of such ligands, but the standard immunological techniques are not always dependable for this purpose.
Quantification of low molecular weight target specific binding ligands generally involves a competition between unlabeled ligand (in a test sample) and labeled ligand for the binding sites of a receptor for the ligand. This is a typical competitive specific binding assay. Ligand in the test sample displaces labeled ligand from the receptor, and the amount of displacement is quantified by various methods. Measurement of bound labeled ligand results in an inverse correlation between the amount of ligand in the sample and the generated signal. Measurement of unbound labeled ligand will yield a positive correlation.
Generally, the signal from bound ligand is easier to measure, but the signal is more difficult to interpret by health practitioners where there is a lack of sophisticated diagnostic training and equipment (for example, in doctors' offices).
Another problem with standard competitive specific binding assays is that the measurement of the free or bound labeled ligand is limited by the efficiency of the separation of free and bound molecules. If the free labeled ligand is not successfully separated from the bound labeled ligand, the sensitivity and specificity of the assay will be adversely affected.
Thus, there is a need for a sensitive competitive binding assay in which efficient separation and measurement of free labeled ligand is achieved to rapidly and easily detect low concentrations of various target specific binding ligands (especially those of low molecular weight).
Copending and commonly assigned U.S. Ser. No. 08/250,980, entitled "Specific Binding Assay and Test Kit Using Enzyme Inhibitor and Anti-Inhibitor Antibodies" and filed on May 31, 1994 by Contestable, Snyder, Corona-Howard and Grogan, provides such an assay which solves the noted problems.
However, the assay of that application requires the use of some sophisticated antibodies which are specific to the reporter enzyme used to generate signal. Those antibodies have other critical properties which render the assay advantageous. Such reagents may be difficult to develop or manufacture consistently for certain assays, and it would therefore be desirable to avoid their use if possible while retaining the remarkable advantages provided by the general assay format. Thus, an alternative assay which uses less sophisticated reagents and which is simpler to manufacture and to use is desired.
U.S. Pat. No. 4,859,583 (Heller et al) describes a chemiluminescent immunoassay for low molecular weight antigens using a device having multiple chambers. In the described assay, sample antigen displaces labeled antigen bound to antibody. The labeled antigen diffuses into a second chamber wherein a light signal is generated. There are several problems associated with this approach, but the primary problem is that the assay is necessarily slow since diffusion must occur across a membrane. Moreover, the device is complicated and requires a suitable membrane for each given analyte (antigen). It would be desirable to have a faster and simpler assay for low molecular weight analytes.